Identification of free-B-ring flavonoids as potent COX-2 inhibitors

ABSTRACT

The present invention provides a novel method for inhibiting the cyclooxygenase COX-2. The method is comprised of administering a composition containing a Free-enzyme B-Ring flavonoid or a composition containing a mixture of Free-B-Ring flavonoids to a host in need thereof. The present also includes novel methods for the prevention and treatment of COX-2 mediated diseases and conditions. The method for preventing and treating COX-2 mediated diseases and conditions is comprised of administering to a host in need thereof an effective amount of a composition containing a Free-B-Ring flavonoid or a composition containing a mixture of Free-B-Ring flavonoids and a pharmaceutically acceptable carrier.

FIELD OF THE INVENTION

[0001] This invention relates generally to a method for the preventionand treatment of COX-2 mediated diseases and conditions. Specifically,the present invention relates to a method for the prevention andtreatment of COX-2 mediated diseases and conditions by administration ofcompounds referred to herein as Free-B-Ring flavonoids. Included in thisinvention is an improved method to generate standardized Free-B-Ringflavonoid extracts from plant sources.

BACKGROUND OF THE INVENTION

[0002] The liberation and metabolism of arachidonic acid (AA) from thecell membrane, results in the generation of pro-inflammatory metabolitesby several different pathways. Arguably, the two most important pathwaysto inflammation are mediated by the enzymes 5-lipoxygenase (5-LO) andcyclooxygenase (COX). These are parallel pathways that result in thegeneration of leukotrienes and prostaglandins, respectively, which playimportant roles in the initiation and progression of the inflammatoryresponse. These vasoactive compounds are chemotaxins, which both promoteinfiltration of inflammatory cells into tissues and serve to prolong theinflammatory response. Consequently, the enzymes responsible forgenerating these mediators of inflammation have become the targets formany new anti-inflammatory drugs.

[0003] Inhibition of the enzyme cyclooxygenase (COX) is the mechanism ofaction attributed to most nonsteroidal anti-inflammatory drugs (NSAIDS).There are two distinct isoforms of the COX enzyme (COX-1 and COX-2) thatshare approximately 60% sequence homology, but differ in expressionprofiles and function. COX-1 is a constitutive form of the enzyme thathas been linked to the production of physiologically importantprostaglandins, which help regulate normal physiological functions, suchas platelet aggregation, protection of cell function in the stomach andmaintenance of normal kidney function. (Dannhardt and Kiefer (2001) Eur.J. Med. Chem. 36:109-26). The second isoform, COX-2, is a form of theenzyme that is inducible by pro-inflammatory cytokines, such asinterleukin-1β (IL-1β) and other growth factors. (Herschmann (1994)Cancer Metastasis Rev. 134:241-56; Xie et al. (1992) Drugs Dev. Res.25:249-65). This isoform catalyzes the production of prostaglandin E2(PGE2) from arachidonic acid (AA). Inhibition of COX-2 is responsiblefor the anti-inflammatory activities of conventional NSAIDs.

[0004] Because the mechanism of action of COX-2 inhibitors overlaps withthat of most conventional NSAID's, COX-2 inhibitors are used to treatmany of the same symptoms, including pain and swelling associated withinflammation in transient conditions and chronic diseases in whichinflammation plays a critical role. Transient conditions includetreatment of inflammation associated with minor abrasions, sunburn orcontact dermatitis, as well as, the relief of pain associated withtension and migraine headaches and menstrual cramps. Applications tochronic conditions include arthritic diseases, such as rheumatoidarthritis and osteoarthritis. Although, rheumatoid arthritis is largelyan autoimmune disease and osteoarthritis is caused by the degradation ofcartilage in joints, reducing the inflammation associated with eachprovides a significant increase in the quality of life for thosesuffering from these diseases. (Wienberg (2001) Immunol. Res. 22:319-41;Wollhiem (2000) Curr. Opin. Rheum. 13:193-201). In addition torheumatoid arthritis, inflammation is a component of rheumatic diseasesin general. Therefore, the use of COX inhibitors has been expanded toinclude diseases, such as systemic lupus erythromatosus (SLE) (Goebel etal. (1999) Chem. Res. Tox. 12:488-500; Patrono et al. (1985) J. Clin.Invest. 76:1011-1018), as well as, rheumatic skin conditions, such asscleroderma. COX inhibitors are also used for the relief of inflammatoryskin conditions that are not of rheumatic origin, such as psoriasis, inwhich reducing the inflammation resulting from the over production ofprostaglandins could provide a direct benefit. (Fogh et al. (1993) ActaDerm Venerologica 73:191-3). Simply stated COX inhibitors are useful forthe treatment of symptoms of chronic inflammatory diseases, as well as,the occasional ache and pain resulting from transient inflammation.

[0005] In addition to their use as anti-inflammatory agents, anotherpotential role for COX inhibitors is in the treatment of cancer. Overexpression of COX-2 has been demonstrated in various human malignanciesand inhibitors of COX-2 have been shown to be efficacious in thetreatment of animals with skin, breast and bladder tumors. While themechanism of action is not completely defined, the over expression ofCOX-2 has been shown to inhibit apoptosis and increase the invasivenessof tumorgenic cell types. (Dempke et al. (2001) J. Can. Res. Clin.Oncol. 127:411-17; Moore and Simmons (2000) Current Med. Chem.7:1131-44). It is possible that enhanced production of prostaglandinsresulting from the over expression of COX-2 promotes cellularproliferation and consequently, increases angiogenesis. (Moore andSimmons (2000) Current Med. Chem. 7:1131-44; Fenton et al. (2001) Am. J.Clin. Oncol. 24:453-57).

[0006] There have been a number of clinical studies evaluating COX-2inhibitors for potential use in the prevention and treatment ofdifferent type of cancers. Aspirin, a non-specific NSAID, for example,has been found to reduce the incidence of colorectal cancer by 40-50%(Giovannucci et al. (1995) N Engl J Med. 333:609-614) and mortality by50% (Smalley et al. (1999) Arch Intern Med. 159:161-166). In 1999, theFDA approved the COX-2 inhibitor CeleCOXib for use in FAP (FamilialAdemonatous Polyposis) to reduce colorectal cancer mortality. It isbelieved that other cancers, with evidence of COX-2 involvement, may besuccessfully prevented and/or treated with COX-2 inhibitors including,but not limited to esophageal cancer, head and neck cancer, breastcancer, bladder cancer, cervical cancer, prostate cancer, hepatocellularcarcinoma and non-small cell lung cancer. (Jaeckel et al. (2001) Arch.Otolarnygol. 127:1253-59; Kirschenbaum et al. (2001) Urology 58:127-31;Dannhardt and Kiefer (2001) Eur. J. Med. Chem. 36:109-26). COX-2inhibitors may also prove successful in preventing colon cancer inhigh-risk patients. There is also evidence that COX-2 inhibitors canprevent or even reverse several types of life-threatening cancers. Todate, as many as fifty studies show that COX-2 inhibitors can preventpremalignant and malignant tumors in animals, and possibly preventbladder, esophageal and skin cancers as well.

[0007] Recent scientific progress has identified correlations betweenCOX-2 expression, general inflammation and the pathogenesis ofAlzheimer's Disease (AD). (Ho et al. (2001) Arch. Neurol. 58:487-92). Inanimal models, transgenic mice that over express the COX-2 enzyme haveneurons that are more susceptible to damage. The National Institute onAging (NIA) is launching a clinical trial to determine whether NSAIDscan slow the progression of Alzheimer's Disease. Naproxen (anon-selective NSAID) and rofeCOXib (Vioxx, a COX-2 specific selectiveNSAID) will be evaluated. Previous evidence has indicated inflammationcontributes to Alzheimer's Disease. According to the Alzheimer'sAssociation and the NIA, about 4 million people suffer from AD in theU.S.; and this is expected to increase to 14 million by mid-century.

[0008] The COX enzyme (also known as prostaglandin H2 synthase)catalyzes two separate reactions. In the first reaction, arachidonicacid is metabolized to form the unstable prostaglandin G2 (PGG2), acyclooxygenase reaction. In the second reaction, PGG2 is converted tothe endoperoxide PGH2, a peroxidase reaction. The short-lived PGH2non-enzymatically degrades to PGE2. The compounds described herein arethe result of a discovery strategy that combined an assay focused on theinhibition of COX-1 and COX-2 peroxidase activity with a chemicaldereplication process to identify novel inhibitors of the COX enzymes.

[0009] Flavonoids are a widely distributed group of natural products.The intake of flavonoids has been demonstrated to be inversely relatedto the risk of incident dementia. The mechanism of action, while notknown, has been speculated as being due to the anti-oxidative effects offlavonoids. (Commenges et al. (2000) Eur. J. Epidemiol 16:357-363).Polyphenol flavones induce programmed cell death, differentiation, andgrowth inhibition in transformed colonocytes by acting at the mRNA levelon genes including COX-2, Nf kappaB and bcl-X(L). (Wenzel et al. (2000)Cancer Res. 60:3823-3831). It has been reported that the number ofhydroxyl groups on the B ring is important in the suppression of COX-2transcriptional activity. (Mutoh et al. (2000) Jnp. J. Cancer Res.91:686-691).

[0010] Free-B-Ring flavones and flavonols are a specific class offlavonoids, which have no substituent groups on the aromatic B ring, asillustrated by the following general structure:

[0011] wherein

[0012] R₂, R₃, R4, and R₅ are independently selected from the groupconsisting of —H, —OH, —SH, OR, —SR, —NH₂, —NHR, —NR₂, —NR₃ ⁺X⁻, acarbon, oxygen, nitrogen or sulfur, glycoside of a single or acombination of multiple sugars including, but not limited toaldopentoses, methyl-aldopentose, aldohexoses, ketohexose and theirchemical derivatives thereof;

[0013] wherein

[0014] R is an alkyl group having between 1-10 carbon atoms; and

[0015] X is selected from the group of pharmaceutically acceptablecounter anions including, but not limited to hydroxyl, chloride, iodide,sulfate, phosphate, acetate, fluoride, carbonate, etc.

[0016] Free-B-Ring flavonoids are relatively rare. Out of a total 9396flavonoids synthesized or isolated from natural sources, only 231Free-B-Ring flavonoids are known. (The Combined Chemical Dictionary,Chapman & Hall/CRC, Version 5:1 June 2001).

[0017] The Chinese medicinal plant, Scutellaria baicalensis containssignificant amounts of Free-B-Ring flavonoids, including baicalein,baicalin, wogonin and baicalenoside. Traditionally, this plant has beenused to treat a number of conditions including clearing away heat,purging fire, dampness-warm and summer fever syndromes; polydipsiaresulting from high fever; carbuncle, sores and other pyogenic skininfections; upper respiratory infections, such as acute tonsillitis,laryngopharyngitis and scarlet fever; viral hepatitis; nephritis;pelvitis; dysentery; hematemesis and epistaxis. This plant has alsotraditionally been used to prevent miscarriage (See Encyclopedia ofChinese Traditional Medicine, ShangHai Science and Technology Press,ShangHai, China, 1998). Clinically Scutellaria is now used to treatconditions such as pediatric pneumonia, pediatric bacterial diarrhea,viral hepatitis, acute gallbladder inflammation, hypertension, topicalacute inflammation, resulting from cuts and surgery, bronchial asthmaand upper respiratory infections (Encyclopedian of Chinese TraditionalMedicine, ShangHai Science and Technology Press, ShangHai, China, 1998).The pharmacological efficacy of Scutellaria roots for treating bronchialasthma is reportedly related to the presence of Free-B-Ring flavonoidsand their suppression of eotaxin associated recruitment of eosinophils.(Nakajima et al. (2001) Planta Med. 67(2):132-135).

[0018] Free-B-Ring flavonoids have been reported to have diversebiological activity. For example, galangin (3,5,7-trihydroxyflavone)acts as anti-oxidant and free radical scavenger and is believed to be apromising candidate for anti-genotoxicity and cancer chemoprevention.(Heo et al. (2001) Mutat. Res. 488(2):135-150). It is an inhibitor oftyrosinase monophenolase (Kubo et al. (2000) Bioorg. Med. Chem.8(7):1749-1755), an inhibitor of rabbit heart carbonyl reductase(Imamura et al. (2000) J. Biochem. 127(4):653-658), has antimicrobialactivity (Afolayan and Meyer (1997) Ethnopharmacol. 57(3):177-181) andantiviral activity (Meyer et al. (1997) J. Ethnopharmacol. 56(2):165-169). Baicalein and galangin, two other Free-B-Ring flavonoids, haveantiproliferative activity against human breast cancer cells. (So et al.(1997) Cancer Lett. 112(2):127-133).

[0019] Typically, flavonoids have been tested for activity randomlybased upon their availability. Occasionally, the requirement ofsubstitution on the B-ring has been emphasized for specific biologicalactivity, such as the B-ring substitution required for high affinitybinding to p-glycoprotein (Boumendjel et al. (2001) Bioorg. Med. Chem.Lett. 11(1):75-77); cardiotonic effect (Itoigawa et al. (1999) J.Ethnopharmacol. 65(3): 267-272), protective effect on endothelial cellsagainst linoleic acid hydroperoxide-induced toxicity (Kaneko and Baba(1999) Biosci Biotechnol. Biochem 63(2):323-328), COX-1 inhibitoryactivity (Wang (2000) Phytomedicine 7:15-19) and prostaglandinendoperoxide synthase (Kalkbrenner et al. (1992) Pharmacology 44(1):1-12). Only a few publications have mentioned the significance of theunsubstituted B ring of the Free-B-Ring flavonoids. One example, is theuse of 2-phenyl flavones, which inhibit NAD(P)H quinone acceptoroxidoreductase, as potential anticoagulants. (Chen et al. (2001)Biochem. Pharmacol. 61(11):1417-1427).

[0020] The reported mechanism of action related to the anti-inflammatoryactivity of various Free-B-Ring flavonoids has been controversial. Theanti-inflammatory activity of the Free-B-Ring flavonoids, chrysin (Lianget al. (2001) FEBS Lett. 496(1):12-18), wogonin (Chi et al. (2001)Biochem. Pharmacol. 61:1195-1203) and halangin (Raso et al. (2001) LifeSci. 68(8):921-93 1), has been associated with the suppression ofinducible cyclooxygenase and nitric oxide synthase via activation ofperoxisome-proliferator activated receptor gamma and influence ondegranulation and AA release. (Tordera et al (1994) Z. Naturforsch [C]49:235-240). It has been reported that oroxylin, baicalein and wogonininhibit 12-lipoxygenase activity without affecting cyclooxygenase. (Youet al. (1999) Arch. Pharm. Res. 22(1):18-24). More recently, theanti-inflammatory activity of wogonin, baicalin and baicalein has beenreported as occurring through inhibition of inducible nitric oxidesynthase and COX-2 gene expression induced by nitric oxide inhibitorsand lipopolysaccharide. (Chen et al. (2001) Biochem. Pharmacol.61(11):1417-1427). It has also been reported that oroxylin acts viasuppression of nuclear factor-kappa B activation. (Chen et al. (2001)Biochem. Pharmacol. 61(11):1417-1427). Finally, wogonin reportedlyinhibits inducible PGE2 production in macrophages. (Wakabayashi andYasui (2000) Eur. J. Pharmacol. 406(3):477-481). Inhibition of thephosphorylation of mitrogen-activated protein kinase and inhibition ofCa²⁺ ionophore A23187 induced prostaglandin E2 release by baicalein hasbeen reported as the mechanism of anti-inflammatory activity ofScutellariae Radix. (Nakahata et al. (1999) Nippon Yakurigaku Zasshi,114, Supp. 11:215P-219P; Nakahata et al. (1998) Am. J. Chin Med.26:311-323). Baicalin from Sculettaria baicalensis, reportedly inhibitssuperantigenic staphylococcal exotoxins stimulated T-cell proliferationand production of IL-1β, interleukin 6 (IL-6), tumor necrosis factor-α(TNF-α), and interferon-γ (IFN-γ). (Krakauer et al. (2001) FEBS Lett.500:52-55). Thus, the anti-inflammatory activity of baicalin has beenassociated with inhibiting the proinflammatory cytokines mediatedsignaling pathways activated by superantigens. However, it has also beenproposed that the anti-inflammatory activity of baicalin is due to thebinding of a variety of chemokines, which limits their biologicalactivity. (Li et al. (2000) Immunopharmacology 49:295-306). Recently,the effects of baicalin on adhesion molecule expression induced bythrombin and thrombin receptor agonist peptide (Kimura et al. (2001)Planta Med. 67:331-334), as well as, the inhibition of mitogen-activatedprotein kinase cascade (MAPK) (Nakahata et al. (1999) Nippon YakurigakuZasshi, 114, Supp 11:215P-219P; Nakahata et al. (1998) Am. J. Chin Med.26:311-323) have been reported. To date there have been no reports thatlink Free-B-Ring flavonoids with COX-2 inhibitory activity.

[0021] To date, a number of naturally occurring Free-B-Ring flavonoidshave been commercialized for varying uses. For example, liposomeformulations of Scutellaria extracts have been utilized for skin care(U.S. Pat. Nos. 5,643,598; 5,443,983). Baicalin has been used forpreventing cancer, due to its inhibitory effects on oncogenes (U.S. Pat.No. 6,290,995). Baicalin and other compounds have been used asantiviral, antibacterial and immunomodulating agents (U.S. Pat. No.6,083,921) and as natural anti-oxidants (Poland Pub. No. 9,849,256).Chrysin has been used for its anxiety reducing properties (U.S. Pat. No.5,756,538). Anti-inflammatory flavonoids are used for the control andtreatment of anorectal and colonic diseases (U.S. Pat. No. 5,858,371),and inhibition of lipoxygenase (U.S. Pat. No. 6,217,875). Flavonoidesters constitute active ingredients for cosmetic compositions (U.S.Pat. No. 6,235,294).

[0022] Japanese Patent No. 63027435, describes the extraction, andenrichment of baicalein and Japanese Patent No. 61050921 describes thepurification of baicalin.

SUMMARY OF THE INVENTION

[0023] The present invention includes methods that are effective ininhibiting the cyclooxygenase enzyme COX-2. The method for inhibitingthe cyclooxygenase enzyme COX-2 is comprised of administering acomposition comprising a Free-B-Ring flavonoid or a compositioncontaining a mixture of Free-B-Ring flavonoids to a host in needthereof.

[0024] The present also includes methods for the prevention andtreatment of COX-2 mediated diseases and conditions. The method forpreventing and treating COX-2 mediated diseases and conditions iscomprised of administering to a host in need thereof an effective amountof a composition comprising a Free-B-Ring flavonoid or a compositioncontaining a mixture of Free-B-Ring flavonoids and a pharmaceuticallyacceptable carrier.

[0025] The Free-B-Ring flavonoids that can be used in accordance withthe following include compounds illustrated by the following generalstructure:

[0026] wherein

[0027] R₁, R₂, R₃, R₄, and R₅ are independently selected from the groupconsisting of —H, —OH, —SH, OR, —SR, —NH₂, —NHR, —NR₂, —NR₃ ⁺X⁻, acarbon, oxygen, nitrogen or sulfur, glycoside of a single or acombination of multiple sugars including, but not limited toaldopentoses, methyl-aldopentose, aldohexoses, ketohexose and theirchemical derivatives thereof;

[0028] wherein

[0029] R is an alkyl group having between 1-10 carbon atoms; and

[0030] X is selected from the group of pharmaceutically acceptablecounter anions including, but not limited to hydroxyl, chloride, iodide,sulfate, phosphate, acetate, fluoride, carbonate, etc.

[0031] The method of this invention can be used to treat and prevent anumber of COX-2 mediated diseases and conditions including, but notlimited to, osteoarthritis, rheumatoid arthritis, menstrual cramps,systemic lupus erythromatosus, psoriasis, chronic tension headaches,migraine headaches, topical wound and minor inflammatory conditions,inflammatory bowel disease and solid cancers.

[0032] The Free-B-Ring flavonoids of this invention may be obtained bysynthetic methods or extracted from the family of plants including, butnot limited to Annonaceae, Asteraceae, Bignoniaceae, Combretaceae,Compositae, Euphorbiaceae, Labiatae, Lauranceae, Leguminosae, Moraceae,Pinaceae, Pteridaceae, Sinopteridaceae, Ulmaceae and Zingiberacea. TheFree-B-Ring flavonoids can be extracted, concentrated, and purified fromthe following genus of high plants, including but not limited to Desmos,Achyrocline, Oroxylum, Buchenavia, Anaphalis, Cotula, Gnaphalium,Helichrysum, Centaurea, Eupatorium, Baccharis, Sapium, Scutellaria,Molsa, Colebrookea, Stachys, Origanum, Ziziphora, Lindera, Actinodaphne,Acacia, Derris, Glycyrrhiza, Millettia, Pongamia, Tephrosia, Artocarpus,Ficus, Pityrogramma, Notholaena, Pinus, Ulmus and Alpinia.

[0033] The compositions of this invention can be administered by anymethod known to one of ordinary skill in the art. The modes ofadministration include, but are not limited to, enteral (oral)administration, parenteral (intravenous, subcutaneous, andintramuscular) administration and topical application. The method oftreatment according to this invention comprises administering internallyor topically to a patient in need thereof a therapeutically effectiveamount of the individual and/or a mixture of multiple Free-B-Ringflavonoids from a single source or multiple sources that include, butnot limited to, synthetically obtained, naturally occurring, or anycombination thereof.

[0034] This invention includes an improved method for isolating andpurifying Free-B-Ring flavonoids from plants containing these compounds.The method of the present invention comprises: a) extracting the groundbiomass of a plant containing Free-B-Ring flavonoids; b) neutralizingand concentrating said extract; and c) purifying said neutralized andconcentrated extract. In a preferred embodiment of the invention theextract is purified using a method selected from the group consisting ofrecrystallization, precipitation, solvent partition and/orchromatographic separation. The present invention provides acommercially viable process for the isolation and purification ofFree-B-Ring flavonoids having desirable physiological activity.

[0035] The present invention implements a strategy that combines aseries of biomolecular screens with a chemical dereplication process toidentify active plant extracts and the particular compounds within thoseextracts that specifically inhibit COX-2 enzymatic activity andinflammation. A total of 1230 plant extracts were screened for theirability to inhibit the peroxidase activity associated with recombinantCOX-2. This primary screen identified 22 plant extracts that werefurther studied for their ability to specifically and selectivelyinhibit COX-2 in vitro in both cell based and whole blood assays. Thoseextracts that were efficacious in vitro were then tested for theirability to inhibit inflammation in vivo using a both air pouch andtopical ear-swelling models of inflammation when administered bymultiple routes (IP and oral). These studies resulted in the discoveryof botanical extracts that inhibited COX-2 activity and were efficaciousboth in vitro and in vivo. These studies also resulted in theidentification of specific Free-B-Ring flavonoids associated with COX-2inhibition in each of these extracts. Applicant believes that this isfirst report of a correlation between Free-B-Ring flavonoid structureand COX-2 inhibitory activity.

[0036] It is to be understood that both the foregoing generaldescription and the following detailed description are exemplary andexplanatory only and are not restrictive of the invention as claimed.

BRIEF DESCRIPTION OF THE FIGURES

[0037]FIG. 1 depicts graphically the inhibition of COX-1 and COX-2 byHTP fractions from Scutellaria baicaensis. The extracts were preparedand fractionated as described in Examples 1 and 3. The extracts wereexamined for their inhibition of the peroxidase activity of recombinantovine COX-1 (▪) or ovine COX-2 (♦). The data is presented as percent ofuntreated control.

[0038]FIG. 2 depicts the high pressure liquid chromatography (HPLC)chromatograms of Free-B-Ring Flavonoids in organic extracts fromScutellaria lateriflora roots (FIG. 2A), Scutellaria orthocalyx roots(FIG. 2B) and Scutellaria baicaensis roots (FIG. 2C).

[0039]FIG. 3 demonstrates the in vivo efficacy of Free-B-Ring Flavonoidsfrom Scutellaria baicaensis on arachidonic acid induced inflammation.The in vivo efficacy was evaluated based on the ability to inhibitswelling induced by direct application of arachidonic acid as describedin Example 9. The average differences in swelling between the treatedears and control ears are represented in FIG. 3A. FIG. 3B demonstratesthe percent inhibition of each group in comparison to the arachidonicacid treated control.

[0040]FIG. 4 illustrates the in vivo efficacy of Free-B-Ring Flavonoidsisolated from Scutellaria baicalensis on inflammation induced byZymosan. Zymosan was used to elicit a pro-inflammatory response in anair pouch as described in Example 9. Markers of inflammation includinginfiltration of pro-inflammatory cells (FIG. 4A), percent inhibition ofMPO activity with in the air pouch fluid (FIG. 4B), and percentinhibition of TNF-α production (FIG. 4C) were used to evaluate theefficacy and mechanism of action of the anti-inflammatory activity ofthe Free-B-Ring Flavonoids from Scutellaria baicalensis.

DETAILED DESCRIPTION OF THE INVENTION

[0041] Various terms are used herein to refer to aspects of the presentinvention. To aid in the clarification of the description of thecomponents of this invention, the following definitions are provided.

[0042] “Free-B-Ring Flavonoids” as used herein are a specific class offlavonoids, which have no substituent groups on the aromatic B ring, asillustrated by the following general structure:

[0043] wherein

[0044] R₁, R₂, R₃, R₄, and R₅ are independently selected from the groupconsisting of —H, —OH, —SH, OR, —SR, —NH₂, —NHR, —NR₂, —NR₃ ⁺X⁻, acarbon, oxygen, nitrogen or sulfur, glycoside of a single or acombination of multiple sugars including, but not limited toaldopentoses, methyl-aldopentose, aldohexoses, ketohexose and theirchemical derivatives thereof;

[0045] wherein

[0046] R is an alkyl group having between 1-10 carbon atoms; and

[0047] X is selected from the group of pharmaceutically acceptablecounter anions including, but not limited to hydroxyl, chloride, iodide,sulfate, phosphate, acetate, fluoride, carbonate, etc.

[0048] “Therapeutic” as used herein, includes treatment and/orprophylaxis. When used, therapeutic refers to humans, as well as, otheranimals.

[0049] “Pharmaceutically or therapeutically effective dose or amount”refers to a dosage level sufficient to induce a desired biologicalresult. That result may be the alleviation of the signs, symptoms orcauses of a disease or any other desirous alteration of a biologicalsystem.

[0050] A “host” is a living subject, human or animal, into which thecompositions described herein are administered.

[0051] Note, that throughout this application various citations areprovided. Each citation is specifically incorporated herein in itsentirety by reference.

[0052] The present invention includes methods that are effective ininhibiting the cyclooxygenase enzyme COX-2. The method for inhibitingthe cyclooxygenase enzyme COX-2 is comprised of administering acomposition comprising a Free-B-Ring flavonoid or a compositioncontaining a mixture of Free-B-Ring flavonoids to a host in needthereof.

[0053] The present also includes methods for the prevention andtreatment of COX-2 mediated diseases and conditions. The method forpreventing and treating COX-2 mediated diseases and conditions iscomprised of administering to a host in need thereof an effective amountof a composition comprising a Free-B-Ring flavonoid or a compositioncontaining a mixture of Free-B-Ring flavonoids and a pharmaceuticallyacceptable carrier.

[0054] The Free-B-Ring flavonoids that can be used in accordance withthe following include compounds illustrated by the general structure setforth above. The Free-B-Ring flavonoids of this invention may beobtained by synthetic methods or may be isolated from the family ofplants including, but not limited to Annonaceae, Asteraceae,Bignoniaceae, Combretaceae, Compositae, Euphorbiaceae, Labiatae,Lauranceae, Leguminosae, Moraceae, Pinaceae, Pteridaceae,Sinopteridaceae, Ulmaceae, and Zingiberacea. The Free-B-Ring flavonoidscan be extracted, concentrated, and purified from the following genus ofhigh plants, including but not limited to Desmos, Achyrocline, Oroxylum,Buchenavia, Anaphalis, Cotula, Gnaphalium, Helichrysum, Centaurea,Eupatorium, Baccharis, Sapium, Scutellaria, Molsa, Colebrookea, Stachys,Origanum, Ziziphora, Lindera, Actinodaphne, Acacia, Derris, Glycyrrhiza,Millettia, Pongamia, Tephrosia, Artocarpus, Ficus, Pityrogramma,Notholaena, Pinus, Ulmus, and Alpinia.

[0055] The flavonoids can be found in different parts of plants,including but not limited to stems, stem barks, twigs, tubers, roots,root barks, young shoots, seeds, rhizomes, flowers and otherreproductive organs, leaves and other aerial parts.

[0056] In order to identify compounds able to inhibit the COX enzymes anextract library composed of 1230 extracts from 615 medicinal plantscollected from China, India, and other countries was created. A generalmethod for preparing the extracts is described in Example 1, which usesthe Scutellaria species for purposes of illustration. The extractionprocess yields an organic and an aqueous extract for each speciesexamined. The results of the extraction of various Scutellaria speciesare set forth in Table 1. These primary extracts are the source materialused in the preliminary assay to identify inhibitors of thecyclooxygenase enzyme's peroxidase activity, which is one of the mainfunctional activities of cyclooxygenase and is responsible forconverting PGG2 to PGH2 and ultimately PGE2, as described in detailabove. This assay is described in Example 2 and the results are setforth in Table 2. With reference to Table 2, it can be seen that twospecies of Scutellaria and three other plant species, all of whichcontain Free-B-ring flavonoids as common components, showed inhibitoryactivity in the primary screen against the peroxidase activity of COX-2albeit to differing degrees. The COX-2 inhibitory activity is foundpredominantly in the organic extracts, which contain the most of mediumpolarity Free-B-Ring flavonoids.

[0057] The COX-2 inhibitory activity from the primary assay of the crudeextracts has been confirmed by measurement of dose response and IC₅₀(the concentration required to inhibit 50% of the enzyme's activity).The IC₅₀ values are set forth in Table 3. As can be seen in Table 3, inthis assay Scutellaria orthocalyx root extract and Murica nana leafextract were the most efficacious (IC₅₀ ₌6-10 mg/mL). Extracts fromScutellaria sp. that demonstrated the greatest selectivity against COX-2were Scutellaria lateriflora (COX-2 IC₅₀: 30 mg/mL; COX-1 IC₅₀: 80mg/mL). Thus, the primary screen for inhibitors of the COX enzymeidentified five extracts containing Free-B-Ring flavonoids that wereefficacious in vitro and some of which demonstrated specificity for theCOX-2 enzyme.

[0058] In order to efficiently identify active compounds from plantextracts, a high throughput fractionation process was used, as describedin Example 3. Briefly, the active organic and aqueous extracts werefractionated using two different methodologies, respectively. Thefractions were collected in a 96 deep well plate. Each of the fractionswas tested for its ability to inhibit COX activity as per the primaryassay, as described in Example 4. The results are set forth in FIG. 1,which depicts the profile of COX-1 and COX-2 inhibition by various HTPfractions derived from the roots of Scutellaria baicalensis. As can beseen in FIG. 1, the most potent COX inhibitory activity was found in twomajor fractions, E11 and F11. It should be noted that three separate HTPfractions actually exhibit inhibitory activity, suggesting that thereare multiple compounds contributing to the observed inhibitory effectsof the whole extract.

[0059] The separation, purification and identification of the activeFree-B-Ring flavonoids present in the organic extract of Scutellariaorthocalyx is described in Example 5. Using the methodology described inExample 5, baicalein was identified as the major active component in theorganic extract from the roots of Scutellaria orthocalyx. As shown inthe Example 6, several other Free-B-Ring flavonoids have been isolatedand tested for inhibition of COX-1 and COX-2 enzymatic activity. Theresults are set forth in Table 4.

[0060] Example 7 and Table 5 set forth the content and quantity of theFree-B-Ring flavonoids in five active plant extracts from threedifferent species of plants. The Free-B-Ring flavonoids are present inmuch greater amounts in the organic extracts verses the aqueousextracts. This explains why the COX-2 inhibitory activity has usuallyshown up in the organic extracts rather than the aqueous extracts.

[0061] The primary assay described in Example 2 to identify activeextracts is a cell free system utilizing recombinant enzymes. To furtherdemonstrate the biological activity of the active extracts andcompounds, two models that represent cell based in vitro efficacy andanimal based in vivo efficacy were employed. The method used to evaluatein vitro efficacy and selectivity is described in Example 8. Two celllines were selected that could be induced to express primarily COX-1(THP-1 cells) and COX-2 (HOSC cells), respectively. Each cell type wasexamined for the production of PGE2, the primary product of the COXenzymes. The results are set forth in Table 6, which shows that threeorganic extracts from three different species of Scutellaria showedinhibition of both the COX-1 and COX-2 enzymes with a preference for theCOX-1 enzyme. While the use of the THP-1 cell line is important anddemonstrates the ability of the active compounds to cross the cellmembrane, it is an immortalized cell line, therefore evaluation of theefficacy of Free-B-Ring flavonoids based on a more relevant model systemis desirable. As a result, the extracts were also evaluated using wholeblood as the primary source of both COX-1 and COX-2 activity. Thissystem measures the inhibition of the production of PGE2 vs. TXB₂ todifferentiate between COX-2 and COX-1 inhibitory activity, respectively.The results, which are set forth in Table 6 demonstrate that both theCOX-1 and COX-2 enzymes are inhibited by the Free-B-Ring flavonoids fromall three Scutellaria root extracts. The IC₅₀ values suggest that withinthis system all Free-B-Ring flavonoids, except those from Scutellariabaicaensis are more efficacious against COX-2. Taken as a whole, theinhibitory effect of the active compounds within these extracts issignificant and efficacious in both cell free and cell-based systems invitro. Also, no cell toxicity been observed in the testing process.

[0062] Two separate in vivo models were employed to determine whetherthe in vitro efficacy observed from the Free-B-Ring flavonoidstranslated to an ability to inhibit in vivo inflammatory responses. Thetwo models are described in Example 9. The first of these systems wasdesigned to measure inflammation resulting directly from the arachidonicacid metabolism pathway. In this example, mice were treated withFree-B-Ring flavonoids from three Scutellaria species prior to thetopical application of AA to the ear to induce the inflammatoryresponse. The effect of pretreating the animals was then measured by theinhibition of the ear swelling using a micrometer. The Free-B-Ringflavonoids containing extracts from these three Scutellaria speciesdemonstrated varying degrees of efficacy. For example, the Free-B-Ringflavonoids extracted from the roots of Scutellaria baicaensis inhibitedear swelling by 60% in comparison to controls when delivered by bothoral and IP routes as illustrated in FIGS. 3A and B. This is the similarto the degree of inhibition seen with the positive control indomethacin,when delivered IP at a concentration of 5 mg/kg. Free-B-Ring flavonoidsextracted from Scutellaria orthocalyx were efficacious when delivered byIP routes of administration, but had no effect when delivered by oralroutes and finally the Free-B-Ring flavonoids extracted from Scutellarialateriflora showed no effect regardless of the route of administration(data not shown).

[0063] The Free-B-Ring flavonoids isolated from Scutellaria baicaensishave been clearly demonstrated to be the most efficacious againstinflammation induced directly by the arachidonic acid. Therefore, theefficacy of these Free-B-Ring flavonoids was examined using a secondmodel in which multiple mechanisms of inflammation are ultimatelyresponsible for the final effect. This system is therefore more relevantto naturally occurring inflammatory responses. Using this model, a verypotent activator of the complement system is injected into an air pouchcreated on the back of Balb/C mice. This results in a cascade ofinflammatory events including, infiltration of inflammatory cells,activation of COX enzymes, resulting in the release of PGE₂, the enzymemyeloperoxidase (MPO), and production of a very specific profile ofpro-inflammatory cytokines including TNF-α. These studies demonstratedthat even though the Free-B-Ring flavonoids isolated from Scutellariabaicaensis did not inhibit the initial infiltration (chemotacticresponse) of inflammatory cells into the air pouch, they blocked theactivation of those cells. This is evidenced by the lack of MPO excretedinto the extracellular fluid of the pouch and the noted lack ofproduction of TNF-α. The results are set forth in FIG. 4. The datademonstrates that the Free-B-Ring flavonoids are efficacious and helpcontrol an inflammatory response in a model system where multipleinflammatory pathways are active.

[0064] The preparation of products for administration in pharmaceuticalpreparations may be performed by a variety of methods well known tothose skilled in the art. The Free-B-Ring flavonoids may be formulatedas an herb powder in the form of its natural existence; as solventand/or supercritical fluid extracts in different concentrations; asenriched and purified compounds through recrystallization, columnseparation, solvent partition, precipitation and other means, as a pureand/or a mixture containing substantially purified Free-B-Ringflavonoids prepared by synthetic methods.

[0065] Various delivery systems are known in the art and can be used toadminister the therapeutic compositions of the invention, e.g., aqueoussolution, encapsulation in liposomes, microparticles, and microcapsules.

[0066] Therapeutic compositions of the invention may be administeredparenterally by injection, although other effective administrationforms, such as intraarticular injection, inhalant mists, orally activeformulations, transdermal iontophoresis or suppositories are alsoenvisioned. One preferred carrier is physiological saline solution, butit is contemplated that other pharmaceutically acceptable carriers mayalso be used. In one preferred embodiment, it is envisioned that thecarrier and Free-B-Ring flavonoid(s) constitute aphysiologically-compatible, slow release formulation. The primarysolvent in such a carrier may be either aqueous or non-aqueous innature. In addition, the carrier may contain otherpharmacologically-acceptable excipients for modifying or maintaining thepH, osmolarity, viscosity, clarity, color, sterility, stability, rate ofdissolution, or odor of the formulation. Similarly, the carrier maycontain still other pharmacologically-acceptable excipients formodifying or maintaining the stability, rate of dissolution, release orabsorption of the ligand. Such excipients are those substances usuallyand customarily employed to formulate dosages for parentaladministration in either unit dose or multi-dose form.

[0067] Once the therapeutic composition has been formulated, it may bestored in sterile vials as a solution, suspension, gel, emulsion, solid,or dehydrated or lyophilized powder; or directly capsulated and/ortableted with other inert carriers for oral administration. Suchformulations may be stored either in a ready to use form or requiringreconstitution immediately prior to administration. The manner ofadministering formulations containing the compositions for systemicdelivery may be via enteral, subcutaneous, intramuscular, intravenous,intranasal or vaginal or rectal suppository.

[0068] The amount of the composition that will be effective in thetreatment of a particular disorder or condition will depend on thenature of the disorder of condition, which can be determined by standardclinical techniques. In addition, in vitro or in vivo assays mayoptionally be employed to help identify optimal dosage ranges. Theprecise dose to be employed in the formulation will also depend on theroute of administration, and the seriousness or advancement of thedisease or condition, and should be decided according to thepractitioner and each patient's circumstances. Effective doses may beextrapolated from dose-response curved derived from in vitro or animalmodel test systems. For example, an effective amount of the compositionof the invention is readily determined by administering graded doses ofthe composition of the invention and observing the desired effect.

[0069] The method of treatment according to this invention comprisesadministering internally or topically to a patient in need thereof atherapeutically effective amount of the individual and/or a mixture ofmultiple Free-B-Ring flavonoids from a single source or multiplesources. The purity of the individual and/or a mixture of multipleFree-B-Ring flavonoids includes, but is not limited to 0.01% to 100%,depending on the methodology used to obtain the compound(s). In apreferred embodiment doses of the Free-B-Ring flavonoids andpharmaceutical compositions containing that same are an efficacious,nontoxic quantity generally selected from the range of 0.01 to 200 mg/kgof body weight. Persons skilled in the art using routine clinicaltesting are able to determine optimum doses for the particular ailmentbeing treated.

[0070] This invention includes an improved method for isolating andpurifying Free-B-Ring flavonoids from plants, which is described inExample 10. In Example 10, Free-B-Ring flavonoids from two Scutellariaspecies were extracted with different solvent systems. The results areset forth in Tables 7 and 8. The improved method of this inventioncomprises: extraction of the ground biomass of a plant containingFree-B-Ring flavonoids with single or combination of organic solventand/or water; neutralization and concentration of the neutralizedextract; and purification of said extract by recrystallization and/orchromatography. As provided above, these Free-B-Ring flavonoids can beisolated from the genera of more than twenty plant families. The methodof this invention can be extended to the isolation of these compoundsfrom any plant source containing these compounds.

[0071] Additionally the Free-B-Ring flavonoids can be isolated fromvarious parts of the plant including, but not limited to, the wholeplant, stems, stem bark, twigs, tubers, flowers, fruit, roots, rootbarks, young shoots, seeds, rhizomes and aerial parts. In a preferredembodiment the Free-B-Ring flavonoids are isolated from the roots,reproductive organs or the whole plant.

[0072] The solvent used for extraction of the ground biomass of theplant includes, but is not limited to water, acidified water, water incombination with miscible hydroxylated organic solvent(s) including, butnot limited to, methanol or ethanol and an mixture of alcohols withother organic solvent(s) such as THF, acetone, ethyl acetate etc. In oneembodiment the extract is neutralized to a pH of 4.5-5.5 and thenconcentrated and dried to yield a powder. The Free-B-Ring flavonoids canthen be purified by various methods including, but not limited torecrystallization, solvent partition, precipitation, sublimation, and/orchromatographic methods including, but not limited to, ion exchangechromatography, absorption chromatography, reverse phase chromatography,size exclusive chromatography, ultra-filtration or a combination ofthereof.

[0073] The following examples are provided for illustrative purposesonly and are not intended to limit the scope of the invention.

EXAMPLES Example 1

[0074] Preparation of Organic and Aquesous from Scutellaria Plants

[0075] Plant material from Scutellaria orthocalyx roots, Scutellariabaicaensis roots or Scutellaria lateriflora whole plant was ground to aparticle size of no larger than 2 mm. Dried ground plant material (60 g)was then transferred to an Erlenmeyer flask and methanol:dichloromethane(1:1) (600 mL) was added. The mixture was shaken for one hour, filteredand the biomass was extracted again with methanol:dichloromethane (1:1)(600 mL). The organic extracts were combined and evaporated under vacuumto provide the organic extract (see Table 1 below). After organicextraction, the biomass was air dried and extracted once with ultra purewater (600 mL). The aqueous solution was filtered and freeze-dried toprovide the aqueous extract (see Table 1 below). TABLE 1 Yield ofOrganic and Aqueous Extracts of various Scutellaria species Plant SourceAmount Organic Extract Aqueous Extract Scutellaria orthocalyx roots 60 g4.04 g 8.95 g Scutellaria baicaensis roots 60 g 9.18 g 7.18 gScutellaria lateriflora 60 g 6.54 g 4.08 g (whole plant)

Example 2

[0076] Inhibition of COX-2 and COX-1 Peroxidase Activity by PlantExtracts from Various Scutellaria Species

[0077] The bioassay directed screening process for the identification ofspecific COX-2 inhibitors was designed to assay the peroxidase activityof the enzyme as described below.

[0078] Peroxidase Assay. The assay to detect inhibitors of COX-2 wasmodified for a high throughput platform (Raz). Briefly, recombinantovine COX-2 (Cayman) in peroxidase buffer (100 mM, TBS, 5 mM EDTA, 1 μMHeme, 0.01 mg epinephrine, 0.094% phenol) was incubated with extract(1:500 dilution) for 15 minutes. Quantablu (Pierce) substrate was addedand allowed to develop for 45 minutes at 25° C. Luminescence was thenread using a Wallac Victor 2 plate reader. The results are set forth inTable 2.

[0079] Table 2 sets forth the inhibition of enzyme by the organic andaqueous extracts obtained from five plant species, including the rootsof two Scutellaria species and extracts from three other plant species,which are comprised of structurally similar Free-B-Ring Flavonoids. Datais presented as the percent of peroxidase activity relative to therecombinant ovine COX-2 enzyme and substrate alone. The percentinhibition by the organic extract ranged from 30% to 90%. TABLE 2Inhibition of COX-2 Peroxidase activity by Scutellaria speciesInhibition of COX-2 Inhibition of COX-2 Plant Source by organic extractby aqueous extract Scutellaria orthocalyx (root) 55% 77% Scutellariabaicaensis (root) 75% 0% Desmodium sambuense 55% 39% (whole plant)Eucaluptus globulus (leaf) 30% 10% Murica nana (leaf) 90% 0%

[0080] Comparison of the relative inhibition of the COX-1 and COX-2isoforms requires the generation of IC₅₀ values for each of theseenzymes. The IC₅₀ is defined as the concentration at which 50%inhibition of enzyme activity in relation to the control is achieved bya particular inhibitor. In the instant case, IC₅₀ values were found torange from 6 to 50 μg/mL and 7 to 80 μg/mL for the COX-2 and COX-1enzymes, respectively, as set forth in Table 3. Comparison, of the IC₅₀values of COX-2 and COX-1 demonstrates the specificity of the organicextracts from various plants for each of these enzymes. The organicextract of Scutellaria lateriflora for example, shows preferentialinhibition of COX-2 over COX-1 with IC₅₀ values of 30 and 80 μg/mL,respectively. While some extracts demonstrate preferential inhibition ofCOX-2, others do not. Examination of the HTP fractions and purifiedcompounds from these fractions is necessary to determine the truespecificity of inhibition for these extracts and compounds. TABLE 3 IC₅₀Values for Human and Ovine COX-2 and COX-1 IC₅₀ Human IC₅₀ Ovine IC₅₀Ovine COX-2 COX-2 Ovine COX-1 Plant Source (μg/mL) (μg/mL) (μg/ml)Scutellaria orthocalyx ND 10 10 (root) Scutellaria baicaensis 30 20 20(root) Scutellaria lateriflora 20 30 80 (whole plant) Eucaluptusglobulus ND 50 50 (leaf) Murica nana 5 6 7 (leaf)

Example 3

[0081] HTP Fractionation of Active Extracts

[0082] Organic extract (400 mg) from Scutellaria baicaensis roots wasloaded onto a prepacked flash column. (2 cm ID×8.2 cm, 10 g silica gel).The column was eluted using a Hitachi high throughput purification (HTP)system with a gradient mobile phase of (A) 50:50 EtOAc:hexane and (B)methanol from 100% A to 100% B in 30 minutes at a flow rate of 5 mL/min.The separation was monitored using a broadband wavelength UV detectorand the fractions were collected in a 96-deep-well plate at 1.9 ml/wellusing a Gilson fraction collector. The sample plate was dried under lowvacuum and centrifugation. DMSO (1.5 mL) was used to dissolve thesamples in each cell and a portion (100 μL was taken for the COXinhibition assay.

[0083] Aqueous extract (750 mg) from Scutellaria baicaensis roots wasdissolved in water (5 mL), filtered through a 1 μm syringe filter andtransferred to a 4 mL HPLC vial. The solution was then injected by anautosampler onto a prepacked reverse phase column (C-18, 15 μm particlesize, 2.5 cm ID×10 cm with precolumn insert). The column was elutedusing a Hitachi high throughput purification (HTP) system with agradient mobile phase of (A) water and (B) methanol from 100% A to 100%B in 20 minutes, followed by 100% methanol for 5 minutes at a flow rateof 10 mL/min. The separation was monitored using a broadband wavelengthUV detector and the fractions were collected in a 96-deep-well plate at1.9 mL/well using a Gilson fraction collector. The sample plate wasfreeze-dried. Ultra pure water (1.5 mL) was used to dissolve samples ineach cell and a portion of 100 μL was taken for the COX inhibitionassay.

Example 4

[0084] Inhibition of COX Peroxidase Activity by HTP Fractions fromVarious Scutellaria Species

[0085] Individual bioactive organic extracts were further characterizedby examining each of the HTP fractions for the ability to inhibit theperoxidase activity of both COX-1 and COX-2 recombinant enzymes. Theresults are depicted in FIG. 1, which depicts the inhibition of COX-2and COX-1 activity by HTP fractions from Scutellaria baicaensis isolatedas described in Example 1 and 3. The profile depicted in FIG. 1 shows apeak of inhibition that is very selective for COX-2. Other Scutellariasp. including Scutellaria orthocalyx and Scutellaria lateriflorademonstrate a similar peak of inhibition (data not shown). However, boththe COX-1 and COX-2 enzymes demonstrate multiple peaks of inhibitionsuggesting that there is more than one molecule contributing to theinitial inhibition profiles.

Example 5

[0086] Isolation and Purification of the Active Free-B-Ring Flavonoidsfrom the Organic Extract of Scutellaria orthocalyx

[0087] The organic extract (5 g) from the roots of Scutellariaorthocalyx, isolated as described in Example 1, was loaded ontoprepacked flash column (120 g silica, 40 μm particle size 32-60 μm, 25cm×4 cm) and eluted with a gradient mobile phase of (A) 50:50EtOAc:hexane and (B) methanol from 100% A to 100% B in 60 minutes at aflow rate of 15 mL/min. The fractions were collected in test tubes at 10mL/fraction. The solvent was evaporated under vacuum and the sample ineach fraction was dissolved in 1 mL of DMSO and an aliquot of 20 μL wastransferred to a 96 well shallow dish plate and tested for COXinhibitory activity. Based on the COX assay results, active fractions#31 to #39 were combined and evaporated. Analysis by HPLC/PDA and LC/MSshowed a major compound with a retention times of 8.9 minutes and a MSpeak at 272 m/e. The product was further purified on a C18semi-preparation column (25 cm×1 cm), with a gradient mobile phase of(A) water and (B) methanol, over a period of 45 minutes at a flow rateof 5 mL/minute. Eighty eight fractions were collected to yield 5.6 mg oflight yellow solid. Purity was determined by HPLC/PDA and LC/MS, andcomparison with standards and NMR data. ¹H NMR: δ ppm. (DMSO-d6) 8.088(2H, m, H-3′,5′), 7.577 (3H, m, H-2′,4′, 6′), 6.932 (1H, s, H-8), 6.613(1H, s, H-3). MS: [M+1]+=271m/e. The compound has been identified asBaicalein. The IC₅₀ of Baicalein against the COX-2 enzyme is 10 μg/mL.

Example 6

[0088] COX Inhibition of Purified Free-B-Ring Flavonoids

[0089] Several other Free-B-Ring Flavonoids have been obtained andtested at a concentration of 20 μg/mL for COX-2 inhibition activitiesusing the methods described above. The results are summarized in Table4. TABLE 4 Inhibition of COX Enzymatic Activity by Purified Free-B-RingFlavonoids Inhibition Inhibition Free-B-Ring Flavonoids of COX-1 ofCOX-2 Baicalein 107% 109% 5,6-Dihydroxy-7-methoxyflavone 75% 59%7,8-Dihydroxyflavone 74% 63% Baicalin 95% 97% Wogonin 16% 12%

Example 7

[0090] HPLC Quantification of Free-B-Ring Flavonoids in Active Extractsfrom Scutellaria orthocalyx Roots, Scutellaria baicaensis Roots andOroxylum indicum Seeds

[0091] The presence and quantity of Free-B-Ring Flavonoids in fiveactive extracts from three different plant species have been confirmedand are set forth in the Table 5. The Free-B-Ring Flavonoids werequantitatively analyzed by HPLC using a Luna C-18 column (250×4.5 mm, 5μm) using 0.1% phosphoric acid and acetonitrile gradient from 80% to 20%in 22 minutes. The Free-B-Ring Flavonoids were detected using a UVdetector at 254 nM and identified based on retention time by comparisonwith Free-B-Ring Flavonoid standards. The HPLC chromatograms aredepicted in FIG. 2. TABLE 5 Free-B-Ring Flavonoid Content in ActivePlant Extracts Total amount % Flavonoids Weight of % Extractible of inActive Extracts Extract from BioMass Flavonoids Extract Scutellariaorthocalyx 8.95 g 14.9% 0.2 mg 0.6% (AE)* Scutellaria orthocalyx 3.43 g5.7% 1.95 mg 6.4% (OE)* Scutellaria baicaensis 7.18 g 12.0% 0.03 mg0.07% (AE)* Scutellaria baicaensis 9.18 g 15.3% 20.3 mg 35.5% (OE)*Oroxylum indicum 6.58 g 11.0% 0.4 mg 2.2% (OE)*

Example 8

[0092] In vitro Study of COX Inhibitory Activity of Free-B-RingFlavonoids from various Scutellaria Species

[0093] In vitro efficacy and COX-2 specificity of Free-B-Ring Flavonoidsisolated from various Scutellaria species were tested in cell basedsystems for their ability to inhibit the generation of arachidonic acidmetabolites. Cell lines HOSC that constitutively express COX-2 and THP-1that express COX-1 were tested for their ability to generateprostaglandin E2 (PGE2) in the presence of arachidonic acid.

[0094] COX-2 Cell Based Assay. HOSC (ATCC#8304-CRL) cells were culturedto 80-90% confluence. The cells were trysinized, washed and resuspendedin 10 mL at 1×10⁶ cells/mL in tissue culture media (MEM). The cellsuspension (200 μL) was plated out in 96 well tissue culture plates andincubated for 2 hours at 37° C. and 5% CO₂. The media was then replacedwith new HOSC media containing 1 ng/mL IL-lb and incubated overnight.The media was removed again and replaced with 190 mL HOSC media. Testcompounds were then added in 10 μL of HOSC media and were incubated for15 minutes at 37° C. Arachidonic acid in HOSC media (20 mL, 100 μM) wasadded and the mixture was incubated for 10 minutes on a shaker at roomtemperature. Supernatant (20 μL) was transferred to new platescontaining 190 μL/well of 100 μM indomethacin in ELISA buffer. Thesupernatants were analyzed as described below by ELISA.

[0095] COX-1 Cell Based Assay. THP-1 cells were suspended to a volume of30 mL (5×10⁵ cells/mL). TPA was added to a final concentration of 10 nMTPA and cultured for 48 hours to differentiate cells to macrophage(adherent). The cells were resuspended in HBSS (25 mL) and added to 96well plates in 200 mL volume at 5×10⁵ cells/well. The test compounds inRPMI 1640 (10 μL) were then added and incubated for 15 minutes at 37° C.Arachidonic acid in RPMI (20 μL) was then added and the mixture wasincubated for 10 minutes on a shaker at room temperature. Supernatant(20 μL) was added to Elisa buffer (190 μL) containing indomethacin (100μM). The supernatants were then analyzed by ELISA, as described below.

[0096] COX-2 Whole Blood assay. Peripheral blood from normal, healthydonors was collected by venipuncture. Whole blood (500 μL) was incubatedwith test compounds and extracts for 15 minutes at 37° C.Lipopolysaccharide (from E. coli serotype 0111:B4) was added to a finalconcentration of 100 μg/mL and cultured overnight at 37° C. Blood wascentrifuged (12,000×g) and the plasma was collected. Plasma (100 μL) wasadded to methanol (400 μL) to precipitate proteins. Supernatants weremeasured for PGE2 production by ELISA. This procedure is a modificationof the methods described by Brideau et al. (1996) Inflamm. Res.45:68-74.

[0097] COX-1 Whole Blood Assay. Fresh blood was collected in tubes notcontaining anti-coagulants and immediately aliquoted into 500 μLaliquots in siliconized microcentrifuge tubes. Test samples were added,vortexed and allowed to clot for 1 hour at 37° C. Samples were thencentrifuged (12,000×g) and the plasma was collected. The plasma (100 μL)was added to methanol (400 μL) to precipitate proteins. Supernatantswere measured for TXB2 production by ELISA. This procedure is amodification of the methods described by Brideau et al. (1996) Inflamm.Res. 45:68-74.

[0098] ELISA Assays. Immunolon-4 ELISA plates were coated with captureantibody 0.5-4 μg/mL in carbonate buffer (pH 9.2) overnight at 4° C. Theplates were washed and incubated for 2 hours with blocking buffer(PBS+1% BSA) at room temperature. The plates were washed again and testsample (100 μL) was added and incubated for 1 hour at room temperaturewhile shaking. Peroxidase conjugated secondary antibody was added in a50 μL volume containing 0.5-4 mg/mL and incubated for 1 hour at roomtemperature while shaking. The plates were then washed three times andTMB substrate (100 μL) was added. The plates were allowed to develop for30 minutes, after which the reaction was stopped by the addition of 1 Mphosphoric acid (100 μL). The plates were then read at 450 nm using aWallac Victor 2 plate reader.

[0099] Cytotoxicity. Cellular cytotoxicity was assessed using acolorimetric kit (Oxford biochemical research) that measures the releaseof lactate dehydrogenase in damaged cells. Assays were completedaccording to manufacturers' directions. No cytotoxicity has beenobserved for any of the tested compounds.

[0100] The results of the assays are set forth in Table 6. The data ispresented as IC₅₀ values for direct comparison. With reference to Table6, IC₅₀ values are generally lower for COX-1 than COX-2. Whole blood wasalso measured for the differential inhibition of PGE2 generation (ameasure of COX-2 in this system) or thromboxane B2 (TXB2) (a measure ofCOX-1 activation). Referring to Table 6, these studies clearlydemonstrate specificity for COX-2 inhibition from the organic extractsin two of the three species tested. Possible reasons for thisdiscrepancy are the fundamental differences between immortalized celllines that constitutively express each of the enzymes and primary cellsderived from whole blood that that are induced to express COX enzymes.Primary cells are the more relevant model to study the in vivoinflammation process. TABLE 6 Inhibition of COX Activity in Whole CellSystems Cell Line Based Assay Whole Blood Assay Plant Source IC₅₀ COX-2IC₅₀ COX-1 IC₅₀ COX-2 IC₅₀ COX-1 Scutellaria orthocalyx 50 μg/mL 18μg/mL 10 μg/mL >50 μg/mL (root) Scutellaria baicaensis 82 μg/mL 40 μg/mL20 μg/mL 8 μg/mL (root) Scutellaria lateriflora 60 μg/mL 30 μg/mL 8μg/mL 20 μg/mL (whole plant)

Example 9

[0101] In vivo Study of COX Inhibitory Activity of Free-B-RingFlavonoids from Various Scutellaria Species

[0102] In vivo inhibition of inflammation was measured using two modelsystems. The first system (ear swelling model) measures inflammationinduced directly by arachidonic acid. This is an excellent measure ofCOX-2 inhibition, but does not measure any of the cellular events whichwould occur upstream of arachidonic acid liberation from cell membranephospholipids by phospholipase A2 (PLA2). Therefore, to determine howinhibitors function in a more biologically relevant response the airpouch model was employed. This model utilizes a strong activator ofcomplement to induce an inflammatory response that is characterized by astrong cellular infiltrate and inflammatory mediator productionincluding cytokines as well as arachidonic acid metabolites.

[0103] Ear Swelling Model. The ear swelling model is a direct measure ofthe inhibition of arachidonic acid metabolism. Arachidonic acid inacetone is applied topically to the ears of mice. The metabolism ofarachidonic acid results in the production of proinflammatory mediatorsproduced by the action of enzymes such as COX-2. Inhibition of theswelling is a direct measure of the inhibition of the enzymes involvedin this pathway. The results are set forth in FIG. 3, which shows theeffects of three extracts delivered either orally by gavage orinterperitoneally (IP) at two time points (24 hours and 1 hour).Free-B-Ring Flavonoids isolated from S. baicaensis inhibited swellingwhen delivered by both IP and gavage although more efficacious by IP.(FIGS. 3A and B). Free-B-Ring Flavonoids isolated from S. orthocalyxinhibited the generation of these metabolites when given IP, but notorally, whereas extracts isolated from S. lateriflora, while beingefficacious in vitro, had no effect in vivo (data not shown).

[0104] Air Pouch Model. Because Free-B-Ring Flavonoids isolated from S.baicaensis were the more efficacious in the ear swelling model, theywere also examined using the air pouch model of inflammation. Briefly,an air pouch was created on the back of the mice by injecting 3 mL ofsterile air. The air pouch was maintained by additional injections of 1mL of sterile air every other day for a period of one week. Animals weredosed using the same methods and concentrations described for theear-swelling model and injected with Zymosan (into the air pouch) toinitiate the inflammatory response. After four hours, the fluid withinthe pouch was collected and measured for the infiltration ofinflammatory cells, myeloperoxidase (MPO) activity (a measure ofcellular activation, degranulation), and tumor necrosis factor-α (TNF-α)production (a measure of activation). The results are set forth in FIG.4.

[0105]FIG. 4A shows the total number of cells collected from the airpouch fluid. While there was a strong response that was inhibited bycontrols (indomethacin), Free-B-Ring Flavonoids isolated from S.baicaensis did not inhibit the infiltration of the inflammatory cells(chemotaxsis). Even though the chemotactic response was not diminished,the fluid was examined to determine whether the infiltrating cells havebecome activated by measuring MPO activity and TNF-α production. FIGS.4B and 4C demonstrate that both MPO activity and TNF-α production aresignificantly reduced when the extract is administered IP, but not bygavages. These data suggest that although the Free-B-Ring Flavonoids donot inhibit the chemotactic response induced by complement activationthey are still effective at reducing inflammation through the preventionof release and production of pro-inflammatory mediators.

[0106] Arachidonic Acid induced ear swelling. The ability of Free-B-RingFlavonoids to directly inhibit inflammation in vivo was measured aspreviously described (Greenspan et al. (1999) J. Med. Chem. 42:164-172;Young et al. (1984) J. Invest. Dermat. 82:367-371). Briefly, groups of 5Balb/C mice were given three dosages of test compounds as set forth inFIG. 4 either interperitoneally (I.P.) or orally by gavage, 24 hours and1 hour prior to the application of arachidonic acid (AA). AA in acetone(2 mg/15 μL) was applied to the left ear, and acetone (15 μL) as anegative control was applied to the right ear. After 1 hour the animalswere sacrificed by CO₂ inhalation and the thickness of the ears wasmeasured using an engineer's micrometer. Controls included animals givenAA, but not treated with anti-inflammatory agents, and animals treatedwith AA and indomethacin (I.P.) at 5 mg/kg.

[0107] Air pouch model of inflammation. Air pouch models were adaptedfrom the methods of Rioja et al. (2000) Eur. J. Pharm. 397:207-217. Airpouches were established in groups of 5 Balb/C mice by injection ofsterile air (3 mL) and maintained by additional injections of 1 mL every2 days for a period of six days. Animals were given three dosages oftest compounds as shown in FIG. 4 either I.P. or orally by gavage, 24hours and 1 hour prior to the injection of 1% Zymosan (1 mL) into thepouch. After 4 hours, the animals were sacrificed by CO₂ inhalation andthe air pouches were lavaged with sterile saline (3 mL). The lavagefluid was centrifuged and the total number of infiltrating cellsdetermined. Supernatants were also retained and analyzed formyleoperoxidase (MPO) activity and the presence of TNF-α by ELISA asmeasures of activation.

Example 10

[0108] Development a Standardized Free-B-Ring Flavonoid Extract fromScutellaria Species

[0109]Scutellaria orthocalyx (500 mg of ground root) was extracted twicewith 25 mL of the following solvent systems. (1) 100% water, (2) 80:20water:methanol, (3) 60:40 water:methanol, (4) 40:60 water:methanol, (5)20:80 water:methanol, (6) 100% methanol, (7) 80:20 methanol:THF, (8)60:40 methanol:THF. The extract solution was combined, concentrated anddried under low vacuum. Identification of chemical components wascarried out by High Pressure Liquid Chromatography using a PhotoDiodeArray detector (HPLC/PDA) and a 250 mm×4.6 mm C18 column. The chemicalcomponents were quantified based on retention time and PDA data usingBaicalein, Baicalin, Scutellarein, and Wogonin standards. The resultsare set forth in Table 7. TABLE 7 Quantification of Free-B-RingFlavonoids Extracted from Scutellaria orthocalyx Using Different SolventSystems Extraction Weight of % Extractible Total amount of % FlavonoidsSolvent Extract from BioMass Flavonoids in Extract 100% water 96 mg19.2% 0.02 mg 0.20% water:methanol 138.3 mg 27.7% 0.38 mg 0.38% (80:20)water:methanol 169.5 mg 33.9% 0.78 mg 8.39% (60:40) water:methanol 142.2mg 28.4% 1.14 mg 11.26% (40:60) water:methanol 104.5 mg 20.9% 0.94 mg7.99% (20:80) 100% methanol 57.5 mg 11.5% 0.99 mg 10.42% methanol:THF59.6 mg 11.9% 0.89 mg 8.76% (80:20) methanol:THF 58.8 mg 11.8% 1.10 mg10.71% (60:40)

[0110]Scutellaria baicaensis (1000 mg of ground root) was extractedtwice using 50 mL of a mixture of methanol and water as follows: (1)100% water, (2) 70:30 water:methanol, (3) 50:50 water:methanol, (4)30:70 water:methanol, (5) 100% methanol. The extract solution wascombined, concentrated and dried under low vacuum. Identification of thechemical components was carried out by HPLC using a PhotoDiode Arraydetector (HPLC/PDA), and a 250 mm×4.6 mm C18 column. The chemicalcomponents were quantified based on retention time and PDA data usingBaicalein, Baicalin, Scutellarein, and Wogonin standards. The resultsare set forth in Table 8. TABLE 8 Quantification of Free-B-RingFlavonoids Extracted from Scutellaria baicaensis Using Different SolventSystems % Extractible Extraction Weight of from Total amount %Flavonoids Solvent Extract BioMass of Flavonoids in Extract 100% water277.5 mg 27.8% 0.01 mg 0.09% water:methanol 338.6 mg 33.9% 1.19 mg11.48% (70:30) water:methanol 304.3 mg 30.4% 1.99 mg 18.93% (50:50)water:methanol 293.9 mg 29.4% 2.29 mg 19.61% (30:70) 100% methanol 204.2mg 20.4% 2.73 mg 24.51%

What is claimed is
 1. A method for inhibiting the cyclooxygenase enzymeCOX-2 comprising administering a composition comprising a Free-B-Ringflavonoid or a composition containing a mixture of Free-B-Ringflavonoids.
 2. The method of claim 1 wherein said Free-B-Ring flavonoidis selected from the group of compounds having the following structure:

wherein R₁, R₂, R₃, R₄, and R₅ are independently selected from the groupconsisting of —H, —OH, —SH, —OR, —SR, —NH₂, —NHR, —NR₂, —NR₃ ⁺X⁻, acarbon, oxygen, nitrogen or sulfur, glycoside of a single or acombination of multiple sugars including, aldopentoses,methyl-aldopentose, aldohexoses, ketohexose and their chemicalderivatives thereof; wherein R is an alkyl group having between 1-10carbon atoms; and X is selected from the group of pharmaceuticallyacceptable counter anions including, hydroxyl, chloride, iodide,sulfate, phosphate, acetate, fluoride and carbonate:
 3. The method ofclaim 1 wherein said Free-B-Ring flavonoid is obtained by organicsynthesis.
 4. The method of claim 1 wherein said Free-B-Ring flavonoidis isolated from a plant part.
 5. The method of claim 4 wherein saidplant is selected from a family consisting of Annonaceae, Asteraceae,Bignoniaceae, Combretaceae, Compositae, Euphorbiaceae, Labiatae,Lauranceae, Leguminosae, Moraceae, Pinaceae, Pteridaceae,Sinopteridaceae, Ulmaceae and Zingiberacea.
 6. The method of claim 4wherein said plant is selected from a genus consisting of Desmos,Achyrocline, Oroxylum, Buchenavia, Anaphalis, Cotula, Gnaphalium,Helichrysum, Centaurea, Eupatorium, Baccharis, Sapium, Scutellaria,Molsa, Colebrookea, Stachys, Origanum, Ziziphora, Lindera, Actinodaphne,Acacia, Derris, Glycyrrhiza, Millettia, Pongamia, Tephrosia, Artocarpus,Ficus, Pityrogramma, Notholaena, Pinus, Ulmus and Alpinia.
 7. The methodof claim 4 wherein the Free-B-Ring flavonoid is isolated from a plantpart selected from the group consisting of stems, stem barks, twigs,tubers, roots, root barks, young shoots, seeds, rhizomes, flowers andother reproductive organs, leaves and other aerial parts.
 8. A methodfor preventing and treating COX-2 mediated diseases and conditionscomprising administering to a host in need thereof an effective amountof a composition comprising a Free-B-Ring flavonoid or a compositioncontaining a mixture of Free-B-Ring flavonoids and a pharmaceuticallyacceptable carrier.
 9. The method of claim 8 wherein said Free-B-Ringflavonoid is selected from the group of compounds having the followingstructure:

wherein R₁, R₂, R₃, R₄, and R₅ are independently selected from the groupconsisting of —H, —OH, —SH, —OR, —SR, —NH₂, —NHR, —NR₂, —NR₃ ⁺X⁻, acarbon, oxygen, nitrogen or sulfur, glycoside of a single or acombination of multiple sugars including, aldopentoses,methyl-aldopentose, aldohexoses, ketohexose and their chemicalderivatives thereof, wherein R is an alkyl group having between 1-10carbon atoms; and X is selected from the group of pharmaceuticallyacceptable counter anions including, hydroxyl, chloride, iodide,sulfate, phosphate, acetate, fluoride and carbonate.
 10. The method ofclaim 8 wherein said Free-B-Ring flavonoid is obtained by organicsynthesis.
 11. The method of claim 8 wherein said Free-B-Ring flavonoidis isolated from a plant part.
 12. The method of claim 11 wherein saidplant is selected from a family consisting of Annonaceae, Asteraceae,Bignoniaceae, Combretaceae, Compositae, Euphorbiaceae, Labiatae,Lauranceae, Leguminosae, Moraceae, Pinaceae, Pteridaceae,Sinopteridaceae, Ulmaceae and Zingiberacea.
 13. The method of claim 11wherein said plant is selected from a genus consisting Desmos,Achyrocline, Oroxylum, Buchenavia, Anaphalis, Cotula, Gnaphalium,Helichrysum, Centaurea, Eupatorium, Baccharis, Sapium, Scutellaria,Molsa, Colebrookea, Stachys, Origanum, Ziziphora, Lindera, Actinodaphne,Acacia, Derris, Glycyrrhiza, Millettia, Pongamia, Tephrosia, Artocarpus,Ficus, Pityrogramma, Notholaena, Pinus, Ulmus and Alpinia.
 14. Themethod of claim 11 wherein the Free-B-Ring flavonoid is isolated fromthe plant part selected from the group consisting of stems, stem barks,twigs, tubers, roots, root barks, young shoots, seeds, rhizomes, flowersand other reproductive organs, leaves and other aerial parts.
 15. Themethod of claim 8 wherein the COX-2 mediated disease or condition isselected from the group consisting of inflammation associated withosteoarthritis, rheumatoid arthritis, menstrual cramps, Systemic LupusErythromatosis, psoriasis, chronic tension headache, migraine headaches,inflammatory bowel disease; topical wound and minor inflammationconditions selected from the group consisting of minor abrasions,sunburn and contact dermatitis; and solid cancers.
 16. The method ofclaim 8 wherein the Free-B-Ring flavonoid composition is comprised of0.01% to 100% of the Free-B-Ring flavanoid.
 17. The method of claim 8wherein the composition is administered in a dosage selected from 0.01to 200 mg/kg of body weight.
 18. The method of claim 8 wherein theroutes of the administration are selected from the group consisting oforal, topical, suppository, intravenous, and intradermic, intragaster,intramusclar, intraperitoneal and intravenous administration in anappropriate pharmaceutical formula.